Project Summary: The long-term goal is to understand the structural and functional mechanisms that control actin cytoskeleton dynamics in health and disease. In its fourth cycle, this grant addresses important gaps of knowledge in previous focus areas, while also expanding into new areas in response to recent developments in the field. Building upon the recent discovery by this lab in collaboration with a group in Norway of actin's dedicated N- terminal acetyltransferase (Naa80), Aim-1 will focus on the mechanism and function of actin N-terminal acetylation (N-acetylation) and isoform variations. Aim-1a will determine the mechanism of N-acetylation, including biochemical and cellular studies of the Naa80-actin interaction and a series of crystal structures of intermediate reaction steps, and using different actin isoforms. Aim-1b will test the hypothesis that myosin activity is strongly dictated by actin isoform variations and N-acetylation. This will resolve an enduring deficiency in the field; most myosin studies have used ?-skeletal actin, overlooking the fact that differences among actin isoforms concentrate at the N-terminus, which is also acetylated and forms part of the myosin- binding site. Aim-1c will explore the role of actin N-acetylation and profilin/actin isoform variations on formin function. Formins are the most important actin filament elongation factors in cells, and this group has found that actin N-acetylation has a profound effect on filament elongation. It is further postulated that actin isoform variations that concentrate at the N-terminus will have an equally strong effect on formin activity. Aim-2 proposes new strategies to tackle persisting questions in the area of actin nucleation, which constitutes a long-standing interest of this lab. Aim-2a will study in parallel Tmods and Lmods, which despite having a common fold have evolved different activities ? pointed-end capping and nucleation, respectively. The source of the functional differences between these two subfamilies will be studied with a focus on Lmod3, implicated in nemaline myopathy. An innovative strategy is proposed to determine the structure of Tmod at the pointed end. It is finally investigated, in vitro and in cells, how specific Tmod and Tropomyosin isoforms interact with each other to assemble morphologically and functionally distinct actin networks. Aim-2b will tackle the long- standing problem of how WASP-family Nucleation Promoting Factors interact with and activate branch formation by the Arp2/3 complex. The plans build upon the ability to perform biochemical and structural studies on the baculovirus-expressed Arp2/3 complex and subcomplexes. Extensive preliminary and published work provide the scientific premise and support feasibility.